Process for the preparation of headgroup-modified phospholipids using phosphatidylhydroxyalkanols as intermediates

ABSTRACT

Phospholipase D enzyme is used to mediate the synthesis of a phosphatidylhydroxyalkanol in a first step. This phosphatidylhydroxyalkanol is reacted to produce a headgroup modified phospholipid in a subsequent step. In the first step, phospholipase D enzyme extract mediates transphosphatidylation of a phospholipid with an alcohol containing at least two hydroxyl groups per molecule, producing reproducible and nearly quantitative yields of a phosphatidylhydroxyalkanol. In the subsequent step, the hydroxyl head group of the phosphatidylhydroxyalkanol is further reacted with amino, carboxylic, halogen or thiol containing molecules to produce a headgroup modified phospholipid.

This is a division of application Ser. No. 08/099,639, filed Jul. 30,1993, now U.S. Pat. No. 5,441,876, to Alok Singh, titled PROCESS FOR THEPREPARATION OF HEADGROUP-MODIFIED PHOSPHOLIPIDS USINGPHOSPHATIDYLHYDROXYALKANOLS AS INTERMEDIATES.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for the preparation ofphospholipids having modified headgroups and, more particularly, to aprocess of using phosphatidylhydroxyalkanols as intermediates in thepreparation of headgroup modified phospholipids.

2. Description of the Related Art

Phospholipids, such as phosphocholines (polymerizable and synthetic),are used to form technologically attractive, stable lipid membranes, theutility of which is well established in fields such as encapsulation andcontrolled release, ion-transport, molecular recognition and biosensors.Synthetic alternations in the phospholipid molecules have not only madeit possible to fabricate molecularly engineered supramolecularassemblies but also to stabilize the resultant microstructures throughpolymerization. Phospholipids have been modified, both in the polarheadgroup region and in the acyl chain region to meet the specific goaldesired for the membrane, including stabilization schemes, site forprotein immobilization, controlled release strategies, molecularrecognition and sensor development. To extend the applicability of lipidmembranes, the charge neutral headgroup of phosphocholines need to bereplaced with reactive functionalities which provide sites for furthersurface modification. The most straightforward routes reported for thesynthesis of such headgroup modified lipids have been the phospholipaseD mediated transphosphatidylation of phosphocholines with substitutedalkanols. Such straightforward routes are not synthetically attractivedue to the low yields of products, and the dependence oftransphosphatidylation on the reaction conditions, chain length ofalkanols as well as the nature of the acyl chains.

For example, in the synthesis of headgroup modified diacetylenicphospholipids by transphosphatidylation, the nature and origin ofphospholipase D enzyme plays an important role. Phospholipase D enzymederived from cabbage exchanges short chain alkanols, but not long chainalkanols, with choline moiety of natural and synthetic phospholipids.Phospholipase D enzyme isolated from rice germ facilitates exchanges ofalkanols independent of their chain length. Phospholipase D enzymeextracted from streptomyces catalyzes the transfer of higher alcoholswith natural phosphocholines, but remains ineffective on syntheticphosphocholines, mixed chain cholines and bulky choline groups.Similarly, pure phospholipase D enzyme from cabbage or peanut does notproduce reproducible yields of diacetylenic phosphatidylethanolamine orphosphatidylbromoethanol.

Alternate synthetic routes involve multi-step, time consuming, low yieldchemical synthesis. Furthermore, the reaction conditions involved inthese alternate synthetic routes are often incompatible with thepolymerizable moieties in lipids which are useful in developing furthertechnological applications.

Thus, there is a need for a convenient, reproducible and high yieldgeneral synthetic route for the preparation of a variety of headgroupmodified phospholipids.

SUMMARY OF THE INVENTION

The present invention overcomes these problems by using phospholipase Denzyme mediated synthesis to produce a phosphatidylhydroxyalkanol in afirst step and chemical reactions the phosphatidylhydroxyalkanol toproduce a headgroup modified phospholipid in a subsequent step. In thefirst step, phospholipase D enzyme extract is used to mediatetransphosphatidylation of a phospholipid with an alcohol containing atleast two hydroxyl groups per molecule, producing reproducible andnearly quantitative yields of a phosphatidylhydroxyalkanol. In thesubsequent step, the hydroxyl headgroup of thephosphatidylhydroxyalkanol is further reacted with amino, carboxylic,halogen or thiol containing molecules to produce a headgroup modifiedphospholipid. The present invention provide a convenient and effectiveprocess for the preparation of headgroup modified phospholipids.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is a two step process for the synthesis of aheadgroup modified phospholipid, comprising:

transphosphatidylating, in the presence of phospholipase D enzyme, aphospholipid with an alcohol containing at least two hydroxyl groups permolecule to form a phosphatidylhydroxyalkanol; and

reacting the phosphatidylhydroxyalkanol with an amino, halogen,carboxylic or thiol containing molecule to form the headgroup modifiedphospholipid.

In the first step, the phospholipase D enzyme may be, for example,isolated from rice germ, extracted from streptomyces, cabbage or peanut.Preferably, the phospholipase D enzyme is extracted from white leaves ofcabbage. The phospholipid is preferably phospholipid1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC₈,9 PC),although the invention may also be applied to modify the headgroups ofother phospholipids. In order to provide a hydroxyl group for furtherreaction in the second step, the alcohol must be at least a diol. Thealcohol may be, for example, a water-soluble diol having primary --OHgroups, such as diethylene glycol, ethylene glycol, 1,3 propanediol,1,4-butanediol, glycerol, and the like.

In the second step, the phosphatidylhydroxyalkanol is used as a reactiveintermediate to produce, for example, esters and halo (Cl, Br or I)analogues of the phosphatidylhydroxyalkanol. Typically, the halogenatingagent is an N,N, 2-trialkyl-1-halopropenylamine. Preferably, thehalogenating agent is an N,N, 2-trimethyl-1-halopropenylamine, an N,N,2-triethyl-1-halopropenylamine or an N,N,2-tributyl-1-halopropenylamine. The halogenating reagent converts thephosphatidylhydroxyalkanol into halogenated analogues in high yields.These halogenated analogues may be further reacted with alkylamines togive aminophospholipids in good yield. These alkylamines used arepreferably primary or secondary, and may be water-soluble or insoluble.Typical alkylamines useful in the present invention include methylamine,dimethylamine, iminodiacetic acid, and the like. Thephosphatidylhydroxyalkanol may also be reacted with maleic anhydride,for example, to produce an ester linked carboxyl terminated lipid.

Moreover, the present invention is also applicable to the synthesis ofpolymerizable phospholipids containing metal chelating iminodiaceticacid functionality in their headgroup region. In this case, the hydroxygroup of the phosphatidylhydroxyalkanol is reacted with, for example asulfuryl halide, such as sulfuryl chloride, to produce a reactive halideintermediate in quantitative yield. This intermediate, upon reactionwith an amine, for example, a primary alcoholic amine such as N,N (biscarboxymethyl) ethanolamine, provides a phospholipid with a headgrouphaving a metal chelating iminodiacetic acid functionality. Furthermore,the method can be extended to increase the linker length between sulfurand nitrogen without going through complex reaction sequence. Theability to control the length and nature of the linkers is advantageousin the study of membrane interactions with biomolecules and ions. Forexample, the hydroxy group of the phosphatidylhydroxyalkanol may bereacted with a dimethyl dihalosilane, --SiX₂ (CH₃)₂ (where X is Cl, I orBr) to form a reactive intermediate and control the length of thelinker. Either of these reactive intermediates (from sulfuryl halide ordimethylhalosilane) may also be reacted, for example, with any primaryalcohol, R--OH (water-soluble or water-insoluble), includingsaccharides. In the reaction of the reactive halide intermediate with aprimary alcohol, an ether linkage, --OR replaces the halogen, --X, ofthe phospholipid, to form useful phospholipids having etherifiedheadgroups.

The present invention has broad applicability in modifying phospholipidsbecause of a) long shelf-life of phospholipase D from. cabbage extract(one month at -20° C.), b) almost quantitative enzymatic transformationsand c) mild reaction conditions.

Materials and Methods

Phospholipase D was extracted from white leaves of cabbage following theprocedure reported by Eibl and Kovatchev in Methods Enzym., vol. 72, p.632, 1981, which is incorporated herein by reference. The proteincontent in the extract was measured to be 1.7 mg/ml. The enzyme extractwas stored in a freezer and used as such. The extract remained activefor one month.

Polymerizable phospholipids, such as 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC₈,9 PC), have thefollowing formula: ##STR1## wherein, as in all the following formulae,R₁ and R₂ may each be an alkyl group containing at least onepolymerizable group, such as a an acrylic acid ester group, an acrylategroup, a diacetylenic group or a diene functionality and may be the sameor different, n is 0 to 1, m is 2, 3 or 4 and R₃, R₄ and R₅ may eachrepresents an alkyl group containing 1 to 4 carbon atoms and may be thesame or different. For example, the compound wherein R₁ and R₂ are both--(CH₂)₈ --C.tbd.C--C.tbd.C--(CH₂)₉ --CH₃, and R₃₋₅ are all --CH₃ wassynthesized following the procedures reported by Leaver et al., Biochem.Biophys. Acta, vol. 732, p. 210, 1983; Singh, J. Lipid Res., vol. 31, p.1522, 1990; and Gupta et al., Proc. Natl. Acad. Sci. USA, vol. 74, p.4315, 1977, which are incorporated herein by reference.

Ether was dried over calcium chloride. Acetate buffer (pH 5.6)containing 0.2M sodium acetate and 0.08M calcium chloride was used inthe enzyme catalyzed reactions. Ethylene glycol, propane diol,butanediol, ethylene diaminetetraacetic acid (EDTA) were obtained fromAldrich Chemical Company. For efficient transformations, the enzymeextract:buffer ratio was kept at 1.75:1, the volume of ether was keptthree times that of the acetate buffer, and the phospholipidconcentration was maintained at 0.66% of the total volume in thereaction flask.

The course of the phospholipase reaction was monitored by thin layerchromatography on silica gel (Merck) employing two solvent systems;chloroform:methanol:water (65:25:4) (A), and chloroform:methanol:ammonia(25% in water) (65:30:3) (B). Spray reagent phosphomolybdic acid wasmade in the lab according to established procedure and Dragendorff'sreaction was purchased from Sigma Chemical Company.N,N,2-trimethyl-1-chloropropenylamine was synthesized following theprocedures reported by Munyemana et al., Tetrahedron Lett., vol.30, p.3077, 1989 and Haveaux et al., Org. Synth., vol. 59, p. 26, 1980, whichare incorporated herein by reference, in their entireties and for allpurposes. Infrared spectra were obtained using a Perkin-Elmer 1800FT-IR. NMR spectra were obtained in CDCl₃ using a Varian EM 390 orBrucker MSL 360 nuclear magnetic resonance spectrometer. Mass spectralanalysis was carried out by fast atom bombardment (FAB) massspectrometry using a Finnigan triple quadrupole mass spectrometer toinsure both the molecular identity and the absence of calcium ions inthe sample.

The phospholipase reaction is performed at temperatures and pH'ssuitable for the action of the enzyme. Typically, temperatures of about20°-40° C., and pH of about 4 to about 7.5 may be used. Preferably, thetemperature is about 25°-30° C. and the pH is about 5.6-6.5.

The concentration of enzyme in the system may affect the speed of thephospholipase reaction. Generally, the higher the concentration ofenzyme, the faster the reaction. The General Procedure outlined belowuses close to the minimum practical concentration of enzyme that onemight wish to use.

Calcium is essential to the phospholipase reaction used in the presentinvention. The concentration of calcium in the reaction system shouldtherefor be at least about 14 mM

While the phospholipase reaction occurs using phospholipase D derivedfrom any biological source, the phospholipase reaction of the presentinvention will not occur if the pure enzyme, rather than a crudepreparation (i.e., direct extract, without affinity separation) is used.Apparently, the presence of a substance associated with phospholipase D,and found in the biological sources of that enzyme, stabilizes itsactive form.

General Procedure for the Synthesis of Phosphatidylhydroxyalkanol

500 mg (0.55 mmols) of1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC₈,9 PC)was dissolved in 30 mL of ether with gentle warming. A one hundred-foldexcess of an appropriate diol, as discussed below, was added. At 40° C.a translucent solution was obtained. Then, a mixture of thephospholipase D extract (30 mL) and 0.2M aq. acetate (NaOAc--AcOH)buffer (80 mM CaCl₂, pH 5.6) (17 mL) was added. A pinkish colordeveloped. An additional 20 mL ether was added. The reaction mixture wasstirred vigorously at 40° C. The course of the reaction was monitored bythin layer chromatography (TLC) on silica plates using solvent systems Aand B. Phosphatidylhydroxyalkanols were revealed at R_(f) 0.53 ascompared with R_(f) 0.33 for phosphocholine (PC) in solvent A, and insolvent B phosphatidylhydroxyalkanols were revealed at R_(f) 0.69 ascompared with R_(f) 0.17 for PC. The reaction mixture was stirred for 10hours. During this time, the reaction mixture was protected from light.The ether was evaporated under reduced pressure and 100 mL of asaturated aq. EDTA solution (pH 8.5) was added to the remaining aqueousphase. The lipid was extracted thrice with a 2/1 CHCl₃ /CH₃ OH (v/v)mixture. The organic fractions were combined and the solvent wasevaporated under reduced pressure at 45° C. The residue was redissolvedin a minimum amount of CHCl₃ and the purity of the lipid was analyzed bythin layer chromatography (solvent A). The developed plates wereanalyzed using Dragendorff's reagent to monitor the disappearance of thePC. The appearance of the product lipid was monitored withphosphomolybdate reagent and iodine vapor. Any unreacted DC₈,9 PC wasremoved by chromatography on a silica column using the followinggradient: 2 col. volumes CHCl₃ ; 2 col. volumes 19/1 CHCl₃ /MeOH; 3 col.volumes 9/1 CHCl₃ /MeOH. To insure the absence of any ion, the lipidswere dissolved in CHCl₃ and treated with an ion exchange resin (BioradAG 50W-X8). The lipid was then dissolved in a minimum amount of warmCHCl₃ and precipitated at 0° C. with acetone.

Some Examples illustrative of the invention will now be given.

EXAMPLE 1 Synthesis of Phosphatidyldiethyleneglycol ##STR2##

500 mg (0.55 mmol) of DC₈,9 PC was reacted with 5.8 g (55 mmol) ofdiethylene glycol in 50 mL of dry ether and 47 mL enzyme-buffer solution(made by mixing 30 mL of phospholipase D extract and 17 mL of acetatebuffer) according to the above-discussed general procedure. The contentswere stirred at 37° C. in the dark. After workup and chromatographyaccording to the general procedure, phosphatidyldiethyleneglycol wascollected in 25% yield. ¹ H NMR (CDCl₃) δ0.88 (t, 6H, --CH₃), 1.26(sharp singlet merged with multiplet, 44H) and 1.46-1.53 (m, 12H for--(CH₂)--), 2.22-2.29 (m, 12H, --C.tbd.C--CH₂ -- and O--C(O)C₂ --),3.65-3.81 (m, 8H, --CH₂ --O--, --CH₂ ----OH), 4.03 (m center, 4H, --CH₂--O--P--O--CH₂ --) and 5.25 (m, 1H, --CH--O--).

EXAMPLE 2 Synthesis of DC₈,9 Phosphatidylhydroxyethanol ##STR3##

Following the general procedure, 400 mg (0.44 mmol) of DC₈,9 PC wasreacted with 2.83 g (45mmol) ethylene glycol and 50 mL of dry ether inthe presence of 47 mL enzyme-buffer solution (made by mixing 30 ml ofphospholipase D extract and 17 mL of acetate buffer). The contents werestirred at 37° C. After the workup of the general procedure, 390 mgcrude lipid was obtained which after purification afforded 300 mg (yield77%) of pure product. TLC analysis using solvent A revealed thehomogeneity of the compound (R_(f) =0.53). ¹ H NMR (300 MHz, CDCl₃)δ0.88 (t, 6H, --CH₃), 1.22-1.44 (s, 44H) merged with 1.44-1.63 (m, 12H,total 56H, --(CH₂)), 2.21-2.35 (m, 12H, --C.tbd.C--CH₂ -- and O--C(O)CH₂--), 3.75 (s, 2H, --CH₂ --OH), 3.99 (s, 4H, --CH₂ --O) 4.12-4.26 (m, 1H,--H--CH--O--), 4.34 (dd, J=4.2 and 11.9 Hz, 1H, --H--CH--O--), and 5.25(p, 1H, --CH--O--). Negative ion mass spectra produced parent ion peakat 871.3 (M-1).

EXAMPLE 3 Synthesis of DC₈,9 Phosphatidylhydroxypropanol ##STR4##

Following the general procedure, 500 mg of DC₈,9 PC in 50 mL of dryether was reacted with 3.95 mL of 1,3 propanediol in the presence of 47mL enzyme-buffer solution (17 mL acetate buffer added to 30 mLphospholipase D extract). Upon workup and acetone precipitation of thegeneral procedure, a quantitative yield of DC₈,9phosphatidylhydroxypropanol was obtained. ¹ H NMR (300 MHz, CDCl₃) δ0.88(t, 6H, --CH₃), 1.2-1.44 (m with sharp singlet, 46H) and 1.44-1.63 (m,12H, total 58H, --(CH₂)--), 2.21-2.35 (m, 12H, --C.tbd.C--CH₂ -- andO--C(O)CH₂ --), 3.70 (s, 2--CH₂ --OH), 4.04-4.3 (broad m, 6H, --CH₂ --O)and 5.25 (m, 1H, --CH--O--).

EXAMPLE 4 Synthesis of DC₈,9 Phosphatidylhydroxybutanol ##STR5##

Following the general procedure, 500 mg of DC₈,9 PC in 50 mL dry etherwas reacted with 4.84 mL of 1,4-butanediol in the presence of 30 mLphospholipase D extract diluted with 17 mL acetate buffer. Workupfollowed by acetone precipitation of the general procedure provided aquantitative yield of DC₈,9 phosphatidylhydroxybutanol. ¹ H NMR (300MHz, CDCl₃) δ0.88 (t, 6H, --CH₃), 1.2-1.44 (m with a sharp singlet, 48H)and 1.44-1.63 (m, 12H, total 60H, --(CH₂)--), 2.24 and 2.33 (t center,J=6.9 Hz, 8H and J=7.1 Hz, 4H for --C.tbd.C--CH₂ -- and O--C(O)CH₂ --resp.), 3.71 (t, 2H, --CH₂ --OH), 4.04-4.21 (m, 4H, --CH₂ --O), 4.36 (d,2H, J=11.9 Hz, 1H, --H--CH--O--) and 5.25 (m, 1H, --CH--O--).

EXAMPLE 5 Synthesis of DC₈,9 Phosphatidylglycerol ##STR6##

Following the general procedure, 538 mg (0.59 mmol) of DC₈,9 PC wasreacted with 5.31 g (59 mmol) glycerol dissolved in 48 mL ether in thepresence of 47 mL enzyme-buffer solution. After workup andchromatography on a silica gel column following the general procedure,290 mg (54% yield) lipid DC₈,9 phosphatidylglycerol was obtained as alight yellow wax.

EXAMPLE 6 Synthesis of DC₈,9 Phosphatidyl-2-chloroalkanols ##STR7##

Synthesis of chloroalkanols was carried out by reacting DC₈,9phosphatidylhydroxyethanol of Example 2 and DC₈,9phosphatidylhydroxybutanol of Example 4 with1-chloro-N,N,2-trimethylpropenylamine in chloroform-d.1-chloro-N,N,2-trimethylpropenylamine has the following formula:##STR8## which can be made, for example, according to the processesdisclosed in Munyemana et al., Tetrahedron Lett., vol.30, p. 3077, 1989and Haveaux et al., Org. Synth., vol. 59, p. 26, 1980. The course of thereaction was followed by NMR. In the case of DC₈,9phosphatidylhydroxyethanol, disappearance of chemical shift at δ3.75(--CH₂ --OH) and appearance at δ3.66 (--CH₂ --Cl) was observed. In thecase of DC₈,9 phosphatidylhydroxybutanol, the ratio of chemical shiftsdue to --CH₂ --OH (δ3.71) and --CH₂ --Cl (δ3.59) was measured to monitorthe course of the reaction. In both the cases, reaction was found to becomplete in 30 minutes. TLC analysis using solvent A revealed thecomplete absence of DC₈,9 phosphatidylhydroxyethanol or DC₈,9phosphatidylhydroxybutanol (R_(f) of 0.53 DC₈,9phosphatidyl-2-chloroalkanols is 0.61).

EXAMPLE 7 Synthesis of DC₈,9 Phosphatidyl-2-chloroethanol ##STR9##

Phospholipid DC₈,9 phosphatidylhydroxyethanol of Example 2 (400 mg, 0.46mmol) was reacted with 1-chloro-N,N,2-trimethylpropenylamine (400 mg, 3mmol) in 4 mL of freshly distilled chloroform (distilled over P₂ O₅).The reaction mixture was carried out at room temperature under nitrogen.The course of reaction was monitored by TLC using solvent A. Aftercompletion of the reaction, the excess chloroform was removed and theresidue was chromatographed on a column of silica gel. Elution withchloroform-methanol (9:1) provided 374 mg DC₈,9phosphatidyl-2-chloroethanol as white wax in 91% yield. ¹ H NMR (300MHz, CDCl₃) δ0.88 (t, 6H, --CH₃), 1.22-1.44 (s, 44H) merged with1.44-1.63 (m, 12H, total 56H, --(CH₂)--), 2.21-2.35 (m, 12H,--C.tbd.C--CH₂ -- and O--C(O)CH₂ --), 3.66 (s, 2H, --CH₂ -- Cl), 3.99(s, 4H, --CH₂ O), 4.12-4.44 (m, 2H, --H--CH--O--), and 5.25 (m,1H--O--).

EXAMPLE 8 Synthesis of DC₈,9 Phosphatidyl-2-(hydroxyethyl)-maleic acid##STR10##

49.0 mg of DC₈,9 phosphatidylhydroxyethanol of Example 2 (0.056 mmol)was reacted with 40 mg (0.4 mmol) maleic anhydride in 1 mL pyridine.Maleic anhydride has the formula ##STR11##

After stirring at room temperature for overnight most of the DC₈,9phosphatidylhydroxyethanol was found consumed with an emerging new spotat lower R_(f) on TLC plate (solvent A). The product was dissolved in2:1 chloroform/methanol and the pyridine was removed by washing with 10%aq. copper sulfate. After removing all the pyridine, the lipid solutionwas washed with 2% hydrochloric acid and the solvent was removed. Theresidue was chromatographed on a column of silica gel to afford 23 mg ofDC₈,9 phosphatidyl-2-(hydroxyethyl)-maleic acid (42% yield). ¹ H NMR(300 MHz, CDCl₃) δ0.88 (t, 6H, --CH₃), 1.22-1.44 (s, 44H) merged with1.44-1.63 (m, 12H, total 56H, --(CH₂)--), 2.21-2.35 (m, 14H,--C.tbd.C--CH₂ -- and O--C(O)CH₂ --), 4.02-4.45 (m, 6H, --CH₂ --O--),5.25 (m, 1H, --CH--O--), 6.28 (d, J=12.6 Hz, 1H, --CH═C--), and 6.4 (d,J=12.6 Hz, 1H, --C═CH--).

EXAMPLE 9 Synthesis of DC₈,9 Phosphatidyl-N-methylaminoethanol,##STR12##

In a teflon capped reaction tube, 30 mg (0.034 mmol) of DC₈,9phosphatidyl-2-chloroethanol from Example 7 dissolved in methylenechloride was reacted with excess of dry methylamine (NH₂ Me) dissolvedin methylene chloride. The contents were stirred at room temperature ina tightly closed reaction tube. TLC analysis using solvent system Arevealed the disappearance of DC₈,9 phosphatidyl-2-chloroethanol andemergence of a new spot at R_(f) 0.40 within three hours due to DC₈,9phosphatidyl-N-methylaminoethanol. After 4 hours of standing at roomtemperature TLC analysis revealed the appearance of a slow moving spot.The reaction was stopped by removing solvent and methylamine by rotaryevaporation. The mixture was separated on a column of silica gel(elution with chloroform-methanol, 9:1). During workup andchromatography steps some hydrolysis was observed. Due to this reasonvariable yields were obtained with the minimum yield being 30%. The slowmoving spot was identified as the lyso analogue by NMR. ¹ H NMR (300MHz, CDCl₃) δ0.88 (t, 6H, --CH₃), 1.22-1.44 (s, 44H) merged with1.44-1.63 (m, 12H, total 56H, --(CH₂)--), 2.21-2.35 (m, 12H,--C.tbd.C--CH₂ -- and O--C(O)CH₂ --), 3.01 (s, 3H, CH₃ --N), 3.43 (s,2H, --CH₂ --N), 3.99 (s, 4H, --CH₂ --O), 4.12-4.40 (m, 2H,--H--CH--O--), and 5.25 (p, 1H, --CH--O--).

EXAMPLE 10 Synthesis of DC₈,9 Phosphatidyl-N,N-dimethylaminoethanol##STR13##

In a teflon capped reaction tube, 30 mg (0.034 mmol) of DC₈,9phosphatidyl-2-chloroethanol dissolved in methylene chloride was reactedwith an excess of dry dimethylamine (HNMe₂) dissolved in methylenechloride. The reaction mixture was tightly closed in a reaction tube andstirred at room temperature. Within one hour, TLC analysis (solventsystem A) revealed the disappearance of DC₈,9Phosphatidyl-2-chloroethanol and emergence of a new spot at R_(f) 0.57due to DC₈,9 phosphatidyl-N,N-dimethylaminoethanol. After 4 hours ofstanding at room temperature TLC revealed the appearance of a spotmoving at R_(f) 0.25 (lyso analogue). The reaction products wereseparated on a column of silica gel (elution with chloroform-methanol,9:1). ¹ H NMR (300 MHz, CDCl₃) δ0.88 (t, 6H, --CH₃), 1.22-1.44 (s, 44H)merged with 1.44-1.63 (m, 12H, total 56H, --(CH₂)--), 2.21-2.35 (m, 12H,--C.tbd.C--CH₂ -- and O--C(O)CH₂ --), 2.94 (s, 3H, --CH.sub. --N), 3.01(s, 3H, CH₃ --N), 3.43 (s, 2H, --CH₂ --N), 3.99 (s, 4H, --CH₂ --O),4.12-4.40 (m, 2H, --H--CH--O--), and 5.25 (p, 1H, --CH--O--). Negativeion mass spectrum revealed parent ion peak at 898.5 (M-1).

EXAMPLE 11 Synthesis of DC₈,9 Phosphatidylethanol-2-chlorosulfonate##STR14##

DC₈,9 phosphatidylhydroxyethanol from Example 2 (169 mg, 0.19 mmol) wasreacted with a ten-fold excess of sulfuryl chloride (SO₂ Cl₂) inchloroform at room temperature. The HCl generated during the reactionmixture was removed by a gentle stream of dry nitrogen bubbled throughthe solution. The completion of the reaction was confirmed by TLC. TLCplates developed with chloroform:methanol:water (65:25:4) revealed anR_(f) of 0.67, which was higher than that of DC₈,9phosphatidylhydroxyethanol (0.50). The reaction was found complete intwo hours. The solvent and the excess sulfuryl chloride was removedunder vacuum to give DC₈,9 phosphatidylethanol-2-chlorosulfonate NMR(CDCl₃) δ_(ppm) 0.88 (t, 6H, CH₃), 1.25 (m with emerging s, 44H,--(CH₂)--), 1.71 (m center, 12H, --CH₂ --CH₂ --COO, and --CH₂ --CH₂--C.tbd.C--), 2.24-2.50 (m center, 12H, --CH₂ --COO, and --CH₂--C.tbd.C--), 4.41-4.51 (m, 8H, --OCH₂), and 5.18-5.33 (m center, 1H,--CHO--).

EXAMPLE 12

Step A: Synthesis of N,N (bis carboxymethyl) ethanolamine. ##STR15##

Reaction between 2.66 g (20 mmol) of iminodiacetic acid (IDA) dissolvedin 9 mL, 7N aqueous KOH, and bromoethanol (6.2 g, 49 mmol) was carriedout by stirring the mixture at 20° C. for 72 hours. The reaction wasmonitored by TLC employing methanol:10% ammonium acetate (2:1) as thesolvent system. Starting material IDA showed lower R_(f) (0.28) thanthat of N,N (bis carboxymethyl) ethanolamine (0.4). After removal of thesolvent, the resulting residue was washed with methanol. The methanolsolution contained N,N (bis carboxymethyl) ethanolamine and unreactedbromoethanol. N,N (bis carboxymethyl ) ethanolamine was separated fromthe reaction mixture first by column chromatography using amethanol:ammonium acetate solvent system. The chromatographed productcontained ammonium acetate as a contaminant, which was removed bydissolving the compound in methanol and crystallizing out the salt.Removal of the solvent gave 0.8 g (23% yield) of pure N,N (biscarboxymethyl) ethanolamine. NMR (D₂ O) δ_(ppm) 3.2 (t center, 2H,HO--CH₂ --CH₂ --N), 3.66 (s, 4H, --CH₂ --N--(CH₂)₂), and 3.71 (t center,2H, HO----CH₂ --CH₂ --N). IR 1640 (COO⁻) cm⁻¹.

Step B: Synthesis of1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phospho-(ethanol,N,N biscarboxymethyl,N ethyl) sulfonate. ##STR16##

The chloroform solution of DC₈,9 phosphatidylethanol-2-chlorosulfonatefrom Example 11 was reacted with N,N (bis carboxymethyl) ethanolaminefrom Step A. DC₈,9 phosphatidylethanol-2-chlorosulfonate was used inthis step as such without further purification. The reaction mixture wasstirred at room temperature for 24 hours under nitrogen atmosphere. Atthis time the reaction seemed complete, as indicated by TLC analysis oftwo chromatograms taken at an interval of 2 hours. Chromatography of thecrude reaction product on a silica column, developed withchloroform:methanol:water (65:25:4), afforded 69 mg (36%) of1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phospho-(ethanol,N,N biscarboxymethyl,N ethyl) sulfonate, which was revealed as a single spot onTLC in the same solvent system as used for column chromatography (R_(f)-0.59). NMR (CDCl₃) δ_(ppm) 0.88 (t, 6H, CH₃), 1.25 (br s, 44H,--(CH₂)--), 1.71 (m center, 12H, --CH₂ --CH₂ --COO, and --CH₂ --CH₂--C.tbd.C--), 2.24-2.50 (m center, 12H, --CH₂ --COO, and --CH₂--C.tbd.C--), 3.7 (m center, 4H, --N--(CH₂ --COOH)₂), 3.9-4.6 (m, 8H,--OCH₂), and 5.15-5.41 (m center, 1H, --CHO--). IR (film) 2217 (vw,br),1460 (S═O), 1244 (PO), 1337 (SO) cm⁻¹.

Numerous modifications and adaptations of the present invention will beapparent to those skilled in the art. For example, with respect toExamples 9 and 10, another amino containing compound, such asiminodiacetic acid, may be used instead of methylamine anddimethylamine, respectively. Thus, it is intended by the followingclaims to cover all modifications and adaptations which fall within thetrue spirit and scope of the invention.

What is claimed is:
 1. A method for the preparation of phospholipidshaving modified headgroups, said method comprising the steps of:a)preparing a first mixture of a diacetylenic phospholipid, a crudeextract of phospholipase D from white leaves of cabbage, and an alcoholcontaining at least two primary hydroxyl groups per molecule; b)reacting said first mixture, in the presence of calcium ions, so as toform, by a phospholipase D enzyme-mediated reaction a reacted mixtureincluding a phosphatidylhydroxyalkanol, said calcium ions being presentat a concentration effective to promote said phospholipase Denzyme-mediated formation of said phosphatidylhydroxyalkanol from saiddiacetylenic phospholipid; c) extracting said phosphatidylhydroxyalkanolfrom said reacted mixture; d) preparing a third mixture of saidphosphatidylhydroxyalkanol and maleic anhydride; e) reacting said thirdmixture so as to form a phosphatidyl 2-(hydroxyalkyl)-maleic acid; andf) extracting said phosphatidyl 2-(hydroxyalkyl)-maleic acid from saidthird mixture.
 2. A method for the preparation of phospholipids havingmodified headgroups, said method comprising the steps of:a) preparing afirst mixture of1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine, a crudeextract of phospholipase D from white leaves of cabbage, and ethyleneglycol; b) reacting said first mixture, in the presence of calcium ions,so as to form, by a phospholipase D enzyme-mediated reaction a reactedmixture including phosphatidylhydroxyethanol, said calcium ions beingpresent at a concentration effective to promote said phospholipase Denzyme-mediated formation of said phosphatidylhydroxyalkanol from said1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine; c)extracting said phosphatidylhydroxyethanol from said reacted mixture; d)preparing a third mixture of said phosphatidylhydroxyethanol and maleicanhydride; e) reacting said third mixture so as to form phosphatidyl2-(hydroxyethyl)-maleic acid; and f) extracting said phosphatidyl2-(hydroxyethyl)-maleic acid from said third mixture.